Cryopyrin-associated periodic syndromes (CAPS) that include the autoinflammatory disorders familial cold autoinflammatory syndrome (FACS), Muckle-Wells syndrome (MWS), and neonatal-onset multiple-system inflammatory disease (NOMID) are caused by dominantly inherited missense mutations in the NLRP3 gene. CAPS represent a continuum of disease severity from recurrent episodes of fever and urticarial skin rash to continous systemic inflammation leading to deforming arthropathy and neurological impairment in the absence of autoimmunity or infection. NLRP3 is a member of the NOD-like receptor (NLR) family that is involved in the recognition of microbial and endogenous stimuli and activation of inflammatory pathways. Recent work from several laboratories including our own indicate that NLRP3 is critical for the activation of caspase-1 through its interaction with the adaptor molecule ASC and the formation of the inflammasome, a molecular platform that drives caspase-1 activation and processing and secretion of IL-12 and IL-18. Surprisingly, we found that mast cells (MCs) are the main cellular source of mature IL-12 in the skin of CAPS patients with NLRP3 mutations. Unlike normal skin, the dermis of patients with NLRP3 mutations produced mature IL-12 constitutively which is associated with marked infiltration of acute inflammatory cells. Consistently, we found that MCs express inflammasome components and produce IL- 12 in response to Toll-like receptor (TLR) ligands and ATP or R837. In MCs deficient in NLRP3 or ASC, processing of pro-caspase-1 and IL-12 secretion triggered by LPS and ATP or R837 was abolished. Notably, MCs from mice in which a common disease- associated NLRP3 mutation was introduced into the NLRP3 locus by homologous recombination secrete mature IL-12 in the absence of ATP or R837 stimulation. These results suggest that MCs act as sentinels in the skin for the recognition of environmental stimuli through the activation of the inflammasome and this function is dysregulated in CAPS patients with NLRP3 mutations. The goal of this proposal is to provide a better understanding of the function of MCs for the regulation of IL-12 and inflammation in the skin as well as the pathogenesis of CAPS. Biochemical, genetic, and cellular approaches will be employed to study the function of NLRP3 in MCs. Given the important role of IL-12 and MCs in immunity and inflammatory disease, understanding of the mechanisms involved in caspase-1 activation and IL-12 production via NLRP3 is expected to have a significant impact in the medical field PUBLIC HEALTH RELEVANCE: IL-12 is an important cytokine that has been linked to the pathogenesis of several inflammatory diseases. Recent studies have revealed that NLRP3, a member of the Nod-like receptor (NLR) family, plays a critical role in the production of IL-12 by mediating the formation of the inflammasome that directs activation of caspase-1, the protease that processes pro-IL-12 to mature IL-12. Traditionally, secretion of IL-12 has been associated with phagocytic cells including macrophages. However, we find that mast cells express inflammasome components and produce mature IL-12 via the activation of the NLRP3 inflammasome. Notably, overproduction of IL-12 in the skin of patients with activating NLRP3 mutations is associated with dysregulated production of IL-12 in MCs. Furthermore, MCs appear to be the main cellular source of IL-12 in the dermis of healthy individuals. We will use genetic and functional approaches to understand the regulation and activation of the NLRP3 inflammasome in MCs and the role of MCs in the pathogenesis of autoinflammatory disease caused by NLRP3 mutations.